Yecuris has developed an in vivo genetic selection system (the FRG KO mouse) that permits extensive humanization of murine liver by transplantation with human hepatocytes. This model has many potential commercial applications in drug development and testing, both in the in vivo and in vitro markets, .The mouse, however, is a small rodent and therefore the number of human hepatocytes that can be obtained for in vitro studies is limited to 80-150 million hepatocytes/animal. A larger animal that could be similarly repopulated would provide significant cost savings and economy of scale. Furthermore, the rat is the animal model of choice in many preclinical applications, particularly toxicology. For these reasons, we decided to generate an FRG KO rat, with mutations completely analogous to those in the FRG KO mouse: Fumary lacetoacetate hydrolase (Fah), Rag2 and common gamma chain of the interleukin receptor (Il2rg). Using TALEN and other gene editing technologies, strains of rats bearing independent mutations in each of these genes have been generated. The goals of this SBIR are straightforward. We propose to a) generate a colony of triple mutant immune deficient Fah knockout, FRG KO rats, and b) optimize the conditions for hepatocyte transplantation and repopulation in using the single knock out Fah-/- rats. Success with this phase I project will enable the use of FRG KO rats for the assessment of commercial production of human hepatocytes. Aim 1: Intercross of rats bearing mutations in Fah, Rag2 and Il2rg to create a colony of triple mutant FRG KO rats. 1a) Knockout alleles of all three genes have been generated, as well as homozygous Fah-/-/Rag2-/- and Fah-/- /Il2rg-/- rats. Homozygous rats for each mutant will be intercrossed to generate triple heterozygote male and female breeders. These will be crossed to generate a colony of triple mutant FRG KO rats. 1b) Fertility of male and female FRG KO rats will be confirmed. Aim 2: Complete characterization of the FRG KO Analysis of key phenotypes in the FRGKO rats. 2a) the relevant phenotypes of triple mutant FRG KO rats will be characterized. 2b) Untransplanted FRG KO rats will be subjected to different NTBC withdrawal regimens in order to determine the most aggressive regimen compatible with survival. Liver function and histology will be analyzed to determine the extent of liver damage. Aim 3: Optimization of NTBC cycling in FRG KO rats transplanted with GFP-SS rat hepatocytes. 3a) Once the NTBC cycling has been determined, FRG KO rats will be transplanted with Fah+/+ GFP-SS rat hepatocytes, subjected to variations of the most severe NTBC cycling to allow for engraftment and repopulation of the Fah+/+ hepatocytes. The transplanted animals will be harvested for quantification of liver repopulation. Aim 4 Humanization and characterization of the FRG KO rat. 4a) Using the optimized NTBC cycling regime determined in Aim 3, we will transplant human hepatocytes into the FRG KO rats. Engraftment and repopulation will be evaluated by serum levels of human albumin and appropriate correlations established. 4b) Immunohistochemistry analysis will be performed on the liver, spleen and kidneys of the humanized FRG KO rats.